The goal of this research is to identify the mechanisms by which eccrine glands are formed with the ultimate aim of regenerating eccrine glands in damaged human skin or in vitro generated skin grafts. Although the expression of En-1 in the ectoderm has been implicated in eccrine gland development in the mouse, the overlapping requirement of engrailed-1 activity for the normal development of the ventral limb in which sweat glands are found has precluded separation of a direct role for En-1 in eccrine gland development from an indirect role in specifying ventral identity. Recently we have generated additional evidence that En-1 plays a direct role in eccrine development. We will dissect that function using an inducible transgenic model and a novel in vitro eccrine gland development assay to test the function of En-1 expression in regulating eccrine gland development. The mechanisms of this effect will be dissected by separating direct effects in the epidermis from indirect effects on the inductive properties of the underlying dermis. Successful completion of these aims will identify the function of En-1 and develop a platform for efficient in vitro analysi of eccrine gland development. The endpoint assay in this work is the in vitro development of eccrine glands, and as such provides direct insight to regeneration of eccrine glands in skin equivalents.